Heavy metal chelator

Cadmium, mercury, and methylmercury all elicit alterations in fura-2 fluorescence that are attenuated by the heavy metal chelator TPEN. TPEN is a low-affinity Ca2+-cation chelator for heavy metals that is cell-permeable. When 10 or 30 μM cadmium chloride was added to cells, the elevated fura-2 fluorescence ratio was significantly reduced to basal levels within 10 minutes (ΔRatio decreased by 119.6±2.4% or 109±1.5% (F340/F380) induced by 10 or 30 μM cadmium chloride, respectively). This suggests that the increase in fura-2 fluorescence ratio caused by cadmium chloride is dependent on intracellular heavy metal cations rather than intracellular Ca2+ [1]. TPEN is a metal chelator that uses copper redox cycling to target colon cancer cells. TPEN decreased cell viability in a way that was dependent on both dose and time. Copper redox cycling is also necessary for TPEN-induced cell death since the copper chelator neocuproline prevents DNA damage and lowers the expression of the ATM, γ-H2AX, pChk1, and other proteins. All three of the cell lines exhibit ATM/ATR signaling in response to low TPEN concentrations; however, preincubation with certain ATM and DNA-PK inhibitors prevented TPEN-induced DNA damage from occurring in the cells [2].

Stimulation with heavy metals is known to induce calcium (Ca(2+)) mobilization in many cell types. Interference with the measurement of intracellular Ca(2+) concentration by the heavy metals in cells loaded with Ca(2+) indicator fura-2 is an ongoing problem. In this study, we analyzed the effect of heavy metals on the fura-2 fluorescence ratio in human SH-SY5Y neuroblastoma cells by using TPEN, a specific cell-permeable heavy metal chelator. Manganese chloride (30-300 µM) did not cause significant changes in the fura-2 fluorescence ratio. A high concentration (300 µM) of lead acetate induced a slight elevation in the fura-2 fluorescence ratio. In contrast, stimulation with cadmium chloride, mercury chloride or MeHg (3-30 µM) elicited an apparent elevation of the fura-2 fluorescence ratio in a dose-dependent manner. In cells stimulated with 10 or 30 µM cadmium chloride, the addition of TPEN decreased the elevated fura-2 fluorescence ratio to basal levels. In cells stimulated with mercury or MeHg, the addition of TPEN significantly decreased the elevation of the fura-2 fluorescence ratio induced by lower concentrations (10 µM) of mercury or MeHg, but not by higher concentrations (30 µM). Pretreatment with Ca(2+) channel blockers, such as verapamil, 2-APB or lanthanum chloride, resulted in different effects on the fura-2 fluorescence ratio. Our study provides a characterization of the effects of several heavy metals on the mobilization of divalent cations and the toxicity of heavy metals to neuronal cells.[1]

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Recently, we showed that the metal chelator TPEN targets colon cancer cells through redox cycling of copper. Here, we studied the DNA damage potential of TPEN and deciphered the role of Chk1, ATM and DNA-PK in TPEN-induced toxicity in 3 human colon cancer cell lines, HCT116, SW480 and HT29. We also investigated the role of reactive oxygen species (ROS) in TPEN-induced DNA damage. TPEN reduced cell viability in a dose- and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of γ-H2AX protein and activation of ATM/ATR signaling pathway. Cell death by TPEN was dependent on ROS generation as evidenced by the reversal of cell viability, and DNA damage and the abrogation of γ-H2AX levels in the presence of antioxidants. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10µM). TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression. Cell death by low TPEN concentrations, involved ATM/ATR signaling in all 3 cell lines, since pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells from TPEN-induced DNA damage. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of γ-H2AX and reversed cell death, suggesting that Chk1 and DNA-PK mediate TPEN-induced cytotoxicity in colon cancer cells. This study shows for the first time the involvement of Chk1, DNA-PK and ATM in TPEN-induced DNA damage and confirms our previous findings that ROS generation and the redox cycling of copper in response to TPEN are the main mechanisms by which this compound induces cell death in human colon cancer cells. Inhibition of ATM or DNA-PK did not reverse cytotoxicity at high TPEN concentrations that cause excessive levels of ROS and irreversible cellular damage[2].

[1]. Heavy metal chelator TPEN attenuates fura-2 fluorescence changes induced by cadmium, mercury and methylmercury. J Vet Med Sci. 2016 Jun 1;78(5):761-7.

[2]. Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells. Cancer Biol Ther. 2016 Nov;17(11):1139-1148.

[3]. Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release. J Neurochem. 2000 Nov;75(5):1878-88.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine is an N-substituted diamine that is ethylenediamine in which the four amino hydrogens are replaced by 2-pyridylmethyl groups. It has a role as a chelator, an apoptosis inducer and a copper chelator. It is a member of pyridines, a tertiary amino compound and a N-substituted diamine. It is functionally related to an ethylenediamine.

C26H28N6

424.55

424.237

C, 73.56; H, 6.65; N, 19.80

16858-02-9

5519

Typically exists as Light yellow to brown solids at room temperature

1.2±0.1 g/cm3

542.1±45.0 °C at 760 mmHg

110-112 °C

281.7±28.7 °C

0.0±1.4 mmHg at 25°C

1.632

2.68

0

6

11

32

419

0

N(C([H])([H])C1=C([H])C([H])=C([H])C([H])=N1)(C([H])([H])C1=C([H])C([H])=C([H])C([H])=N1)C([H])([H])C([H])([H])N(C([H])([H])C1=C([H])C([H])=C([H])C([H])=N1)C([H])([H])C1=C([H])C([H])=C([H])C([H])=N1

CVRXLMUYFMERMJ-UHFFFAOYSA-N

InChI=1S/C26H28N6/c1-5-13-27-23(9-1)19-31(20-24-10-2-6-14-28-24)17-18-32(21-25-11-3-7-15-29-25)22-26-12-4-8-16-30-26/h1-16H,17-22H2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)